TUMOR-NECROSIS-FACTOR INCREASES HEPATOCELLULAR GLUTATHIONE BY TRANSCRIPTIONAL REGULATION OF THE HEAVY SUBUNIT CHAIN OF GAMMA-GLUTAMYLCYSTEINE SYNTHETASE
A. Morales et al., TUMOR-NECROSIS-FACTOR INCREASES HEPATOCELLULAR GLUTATHIONE BY TRANSCRIPTIONAL REGULATION OF THE HEAVY SUBUNIT CHAIN OF GAMMA-GLUTAMYLCYSTEINE SYNTHETASE, The Journal of biological chemistry, 272(48), 1997, pp. 30371-30379
Tumor necrosis factor (TNF) is an inflammatory cytokine that causes ce
ll injury by generation of oxidative stress, Since glutathione (GSH) i
s a key cellular antioxidant that detoxifies reactive oxygen species,
the purpose of our work was to examine the regulation of cellular GSH,
the expression of heavy subunit chain of gamma-glutamylcysteine synth
etase (gamma-GCS-HS), and control of intracellular generation of react
ive oxygen species in cultured rat hepatocytes treated with TNF. Expos
ure of cells to TNF (10,000 units/ml) resulted in depletion of cellula
r GSH levels (50-70%) and overproduction of hydrogen peroxide (2-3-fol
d) and lipid peroxidation, However, cells treated with lower doses of
TNF (250-500 units/ml) exhibited increased levels of GSH (60-80% over
control). TNF treatment increased (70-100%) the levels of gamma-GCS-HS
mRNA, the catalytic subunit of the regulating enzyme in GSH biosynthe
sis. Furthermore, intact nuclei isolated from hepatocytes treated with
TNF transcribed the gamma-GCS-HS gene to a greater extent than contro
l cells, indicating that TNF regulates gamma-GCS-HS at the transcripti
onal level. The capacity to synthesize GSH de novo determined in cell-
free extracts incubated with GSH precursors was greater (50-70%) in he
patocytes that were treated with TNF; however, the activity of GSH syn
thetase remained unaltered by TNF treatment indicating that TNF select
ively increased the activity of gamma-GCS. Despite activation of nucle
ar factor-kappa B (NF-kappa B) by TNF, this transcription factor was n
ot required for TNF-induced transcription of gamma-GCS-HS as revealed
by deletion constructs of the gamma-GCS-HS promoter subcloned in a chl
oramphenicol acetyltransferase reporter vector and transfected into He
pG2 cells, In contrast, a construct containing AP-1 like/metal respons
e regulatory elements increased chloramphenicol acetyltransferase acti
vity upon exposure to TNF. Thus, TNF increases hepatocellular GSH leve
ls by transcriptional regulation of gamma-GCS-HS gene, probably throug
h AP-1/metal response element-like binding site(s) in its promoter, wh
ich may constitute a protective mechanism in the control of oxidative
stress induced by inflammatory cytokines.